Euphorbia factor L1 suppresses breast cancer liver metastasis via DDR1-mediated immune infiltration.

Euphorbia factor L1 (EFL1), a lathyrane-type diterpenoid from the medicinal herb Euphorbia lathyris L., has been documented to possess various pharmacologic actives. However, the function of EFL1 on breast cancer is not clear. In this study, we explored the effect and mechanism of EFL1 on breast cancer liver metastasis. Female BALB/c mice were subjected to breast cancer-surgical hepatic implantation (SHI) to establish breast cancer liver metastasis model in vivo. At 10 days post-surgery, mice were administrated with EFL1 once daily for a total of 2 weeks. Serum AST and ALT activities, abdominal circumference, peritoneal fluid, tumor weight and volume were determined to assess liver and mesenteric re-metastasis of breast cancer. H&E staining was used to observe morphology changes in tumor, liver and small intestine tissues. ELISA was applied to observe inflammatory levels. Tumor DDR1 expression and immune infiltration were determined using western blotting, immunohistochemistry and flow cytometer methods. Our results showed that EFL1 administration improved liver function (AST and ALT activities), ascites, liver metastasis and mesenteric re-metastasis in SHI mice. Also, SHI-induced inflammatory cell infiltration and IL-1ß, IL-6, TNF-α generation in ascites were decreased by EFL1 treatment. Mechanism study revealed that EFL1 intervention enhanced the ratios of CD4+ and CD8+ and CD49b+(NK) T lymphocytes and decreased Treg cells through downregulating DDR1 in the tumor of SHI mice. Furthermore, overexpression of DDR1 abolished the anti-liver metastasis effect and pro-immune infiltration action of EFL1 in SHI mice. Together, our findings suggested that EFL1 protects against breast cancer liver metastasis in vivo by targeting DDR1-mediated immune infiltration.

[Chemical ingredient groups B and C in Kansui Radix stir-fried with vinegar affect diversity of gut microbiota in rat model of malignant ascites induced by Walker-256 cells].

This study aims to explore the effects of chemical ingredient groups B and C in Kansui Radix stir-fried with vinegar on the diversity of gut microbiota in the rat model of malignant ascites, identify the key differential microbial taxa, and reveal the biological mechanism of water-expelling effect of the two chemical ingredient groups. The rat model of malignant ascites induced by Walker-256 cells was established, and phenolphthalein was used as the positive drug. The rats were orally administrated with corresponding agents for consecutive 7 days. On day 6, fresh feces samples were collected from the rats, and 16 S rDNA high-throughput sequencing and GC-MS were employed to determine the composition of gut microbiota and the content of short-chain fatty acids, respectively. On day 7, serum and intestinal tissue samples were collected for the determination of related indicators. Compared with the control group, the model group showed decreased feces volume and urine volume(P<0.01), increased volume of ascites and levels of Na~+, K~+, and Cl~- in urine(P<0.01), down-regulated mRNA and protein levels of intestinal AQP8(P<0.01), lowered abundance of beneficial Lactobacillus(P<0.01) while risen abundance of potential pathogenic Lachnospiraceae and Anaeroplasma(P<0.01), and reduced content of short-chain fatty acids(P<0.01). Compared with the model group, administration with chemical ingredient groups B and C alleviated all the above indicators(P<0.01). In conclusion, chemical ingredient groups B and C in Kansui Radix stir-fried with vinegar could alleviate the disordered gut microbiota in rats with malignant ascites to expel water through increasing the abundance of beneficial Lactobacillus and reducing the abundance of harmful Lachnospiraceae and Anaeroplasma. This study can provide a reference for the reasonable clinical application of Kansui Radix stir-fried with vinegar.

[Effect of Kansui Radix stir-fried with vinegar on fecal metabolomics in Walker-256 rats with ascites].

Utilizing metabolomics technology, this study explored the change of fecal endogenous metabolites in Walker-256 rats with malignant ascites after the administration with Kansui Radix(KR) stir-fried with vinegar(VKR), sought the potential biomarkers in feces which were related to the treatment of malignant ascites by VKR and revealed the biological mechanism of water-expelling effect of VKR. Ultra-fast liquid chromatography-quadrupole-time-of-flight mass spectrometry(UFLC-Q-TOF-MS) was employed to detect the feces of rats in all groups. Principle component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) were conducted to achieve pattern recognition. Combining t-test and variable importance in the projection(VIP) enabled the screening of potential biomarkers for the malignant ascites. Metabolic pathway analysis was accomplished with MetaboAnalyst. Correlation analysis was finally conducted integrating the sequencing data of gut microbiota to elucidate the mechanism underlying the water-expelling effect of VKR. The results showed that both KR and VKR could restore the abnormal metabolism of model rats to some extent, with VKR being inferior to KR in the regulation. Eleven potential biomarkers were identified to be correlated with the malignant ascites and five metabolic pathways were then enriched. Four kinds of gut microbiota were significantly related to the potential biomarkers. The water-expelling effect of VKR may be associated with the regulation of phenylalanine metabolism, biosynthesis of phenylalanine, tyrosine and tryptophan, tryptophan metabolism, glycerophospholipid metabolism, and glycosylphosphatidylinositol(GPI)-anchor biosynthesis. This study can provide a scientific basis for comprehensive understandings of the interaction between gut microbiota and host which has relation to the water-expelling effect of VKR and guide the reasonable clinical application of VKR.

Diverse role of gut microbiota on reduction of ascites and intestinal injury in malignant ascites effusion rats treated with Euphorbia kansui stir-fried with vinegar.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia kansui (EK) is the dried root of Euphorbia kansui S.L.Liou ex S.B.Ho. Clinically, processing with vinegar is for reducing toxicity of EK, and EK stir-fried with vinegar (VEK) is used to treat ascites and edema. VEK has been confirmed to reduce ascites by accelerating the promotion of intestinal contents. AIM OF THE STUDY: The study aimed to investigate whether gut microbiota could affect the expelling water retention effects and the intestinal oxidative damage of EK and VEK on malignant ascites effusion (MAE) rats. MATERIALS AND METHODS: Pseudo-germ-free (PGF) MAE rats or probiotic intervented MAE rats were treated with EK/VEK. Related indicators such as serum, ascites, urine, feces, gastrointestinal tissues were analyzed, and the structure of the gut microbiota were also studied. The relationship between gut microbiota and the expelling water retention effects of EK/VEK where then further investigated. RESULTS: VEK reduce the volume of ascites by promoting urine and feces excretion, AQP8 protein and mRNA expression, when comparing with the MAE rats, also VEK could regulate the disordered gut microbiota in MAE rats. Mixed antibiotics could diminish VEK's expelling water retention effects in MAE rats, but increased oxidative damage in intestine. While existence of gut microbiota (especially probiotics) played an important role in the protection of intestines in VEK treated MAE rats. CONCLUSION: VEK had obvious pharmacological effect on MAE and could regulate gut microbiota, but gut microbiota was not a necessary condition for its pharmacological effects. The probiotics played a synergistic role with VEK in the effects of expelling water retention and intestinal protection.

Comparison of the short-chain fatty acids in normal rat faeces after the treatment of Euphorbia kansui, a traditional Chinese medicine for edoema.

Context: As a toxic traditional Chinese medicine for edoema, Euphorbia kansui S.L. Liou ex S.B. Ho (Euphorbiaceae) (EK) stir-fried with vinegar for detoxification was associated with alterations of gut microbiota. However, the evidence of correlation between short-chain fatty acids (SCFAs) and toxicity of EK has not been confirmed.Objective: In order to study the biological basis of detoxification of EK stir-fried with vinegar (VEK), a rapid, sensitive and validated GC-MS method was developed to determine SCFAs in normal rat faeces after given EK and VEK.Materials and methods: Sprague Dawley rats were orally administered 0.5% CMC-Na (control group), EK (EK-treated group) and VEK powder (VEK-treated group) at 680 mg/kg for six consecutive days (eight rats each group). Fresh faeces samples were promptly collected, derivatized and then analyzed by GC-MS.Results: The ranges of LOD and LOQ were within 0.13-1.79 and 0.45-5.95 µg/mL, respectively. The RSD values of intra-day and inter-day precisions were less than 15%. Four SCFAs were generally stable under four storage conditions. The extraction recoveries were ranged from 53.5% to 97.3% with RSD values lower than 15%. The concentrations of four SCFAs in EK and VEK were decreased significantly compared with those not administered (EK-treated, p < 0.01; VEK-treated, p < 0.05 and p < 0.01). After being stir-fried with vinegar, the concentrations were all increased (p < 0.05 and p < 0.01).Discussion and conclusions: The negative correlation between SCFAs and toxicity of EK may provide evidence for biological mechanism and toxic Chinese medicine.

[Effect of chemical ingredients B and C in Kansui stir-baked with vinegar on fecal metabolomics in rats].

To reveal the toxic mechanism of Kansui stir-baked with vinegar(VEK), conducta comparative study on the metabolites of fecal samples of rats before and after being treated with chemical constituents group B and C(VEKB/VEKC) extracted from VEK by metabolomics approach. The fecal samples of each group were analyzed using ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UFLC-Q-TOF-MS). Then the data was processed by principal component analysis(PCA) and partial least square discriminant analysis(OPLS-DA) to screen and identify biomarkers relating to the toxicity of VEK. Besides, t-test was adopted for univariate statistical analysis, so as to study the changes of these biomarkers in drug groups before and after being treated with VEKB/VEKC and explore the effect of VEKB/VEKC on the metabolism of rat feces. Furthermore, the toxic mechanism of VEKB/VEKC was explored based on the results of the metabolic pathway analysis. The results displayed that compared with control group, the metabolism of fecal samples of VEKB and VEKC treated groups show obvious changes, and the VEKB treated group show more significant changes. A total of 16 potential biomarkers and 5 metabolic pathways relating to the toxicity of VEK were found and identified. And the toxicity of VEK might be associated with the disorder of such metabolic pathways as tryptophan metabolism, primary bile acid biosynthesis, amino sugar and nucleotide sugar metabolism, purine metabolism, and degradation of valine, leucine and isoleucine. This study provides a scientific basis for the clinical safety application of VEK.

Euphorbia kansui fry-baked with vinegar modulates gut microbiota and reduces intestinal toxicity in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia kansui (EK), a kind of toxic traditional Chinese medicine (TCM), is used in the treatment of edema, ascites and asthma. EK fry-baked with vinegar (VEK) is regularly used to reduce the toxicity in TCM. Previous studies have confirmed that fry-baking with vinegar could significantly reduce the significant gastrointestinal toxicity of EK. The toxic side-effects of EK are closely associated with intestinal tract, but existing research results could not provide practical measures for detoxification in terms of the biological effects of EK fry-baked with vinegar. AIM OF THE STUDY: This study aimed to investigate the gastrointestinal toxicity of EK and detoxification of VEK through the regulation of gut microbiota. Thirty male Sprague Dawley (SD) rats were randomly divided equally into 3 groups and received by oral gavage 0.5% CMC-Na (C group), EK (EKC group) or VEK (VEKC group) powder at 680 mg/kg for seven consecutive days. RESULTS: The ten toxic components in VEK were reduced significantly compared with those in EK. After fry-baked with vinegar, those side effects associated with VEK were significantly relieved in terms of histopathology and inflammatory injury indices of intestinal tissues, liver function and oxidative damage indices. The toxicity of EK might be highly correlated with Lactobacillus and Blautia genera. In addition, EK fry-baked with vinegar increased the production of short-chain fatty acids (SCFAs), which are regulated by gut microbiota. CONCLUSIONS: The proportion of main probiotics increased and potentially pathogenic bacteria decreased after EK was fry-baked with vinegar. It turned out that effective detoxification could be achieved by fry-baking with vinegar.

The toxicity and efficacy evaluation of different fractions of Kansui fry-baked with vinegar on Walker-256 tumor-bearing malignant ascites effusion rats and normal rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Kansui, the root of Euphorbia kansui S.L.Liou ex S.B.Ho (E.kansui), is a classical traditional Chinese medicine (TCM) with certain toxicity. According to the theory of TCM, kansui fry-baked wtith vinegar (VEK) possesses low toxicity and mild diuretic and purgative efficacy. In clinical practice, it is commonly used for the treatmtablent of ascites and oliguria. The present study aimed to evaluate the toxicity and efficacy of different fractions of VEK and reveal the underlying material basis by employing an animal model of malignant ascites effusion (MAE) in rats. MATERIALS AND METHODSTA: The MAE rats as the model were constructed in SPF male wistar rats by intraperitoneal injection of Walker-256 tumor cells. The MAE rats were used and randomly divided into the control group (normal rats), control groups with different fractions (VEKA, VEKB, VEKC and VEKD), model group (MAE rats), positive control group (model group with furosemide), model groups with different fractions (VEKA, VEKB, VEKC and VEKD). Histopathological observation was used to confirm Walker-256 tumor-bearing organ injuries in rats. For the efficacy evaluation, the ascites and urine volumes, the urinary electrolyte concentrations (Na+, K+ and Cl-) and pH, the ascites levels of pro-inflammatory cytokines (IL-2, IL-6, TNF-α, IFN-γ and VEGF), PRA, the serum levels of Ang II, ALD and ADH, as well as AQP8 protein expression in the gastrointestinal tract were detected. Furthermore, different levels of indicators were measured in the toxicity evaluation of different fractions both on normal and model rats, including serum liver enzymes (AST and ALT), serum oxidative damage parameters (GSH, MDA, LDH and SOD), expressions of inflammatory parameters (NF-κB, ICAM-1 and E-cadherin) and apoptosis signals (caspase-3, -8, -9, Bcl-2 and Bax) in the liver and gastrointestinal tract. RESULTS: Walker-256 tumor-bearing malignant ascites effusion rats showed obvious hepatic and gastrointestinal injuries by histopathological observation. In the efficacy evaluation, model rats treated with VEKB and VEKC showed significant urine increase (VEKB, P < 0.01; VEKC, P < 0.01) and ascites reduction (VEKB, P < 0.01; VEKC, P < 0.01). These two fractions also balanced the concentrations of Na+, K+ and Cl- in urine (VEKB, all P < 0.05; VEKC, all P < 0.05), remarkably decreased urinary pH (VEKB, P < 0.01; VEKC, P < 0.01), and reduced the ascites levels of IL-2, IL-6, TNF-α, IFN-γ and VEGF (VEKB, all P < 0.01; VEKC, all P < 0.01) in the model rats. Moreover, levels of PRA, the serum Ang II, ALD and ADH of model rats were decreased after treated by VEKB and VEKC (VEKB, all P < 0.05; VEKC, all P < 0.05). Meanwhile, the expression of gastrointestinal AQP8 of the model rats was also enhanced after treated by VEKB and VEKC (VEKB, P < 0.01; VEKC, P < 0.01). In the toxicity evaluation, although VEKB and VEKC caused toxic indexes moved to the worse aspects in normal rats, nearly all of these indicators notably improved in the model rats. Additionally, VEKA showed no effect on the indicators, either in the efficacy evaluation or in the toxicity evaluation. And VEKD could significantly improve some indicators (urine volume, concentration of K+ in urine, serum MDA, AI and caspase-9) in MAE rats. CONCLUSIONS: VEKB and VEKC were demonstrated a significant efficacy in treating malignant ascites effusion, which could reduce hepatic and gastrointestinal damage on the model rats but cause the same damage to the normal. These data embody the traditional Chinese medicine application principle: You Gu Wu Yun. And these results will provide reference for the safer and better clinical utilization of kansui.

Quantitative determination, principal component analysis and discriminant analysis of eight marker compounds in crude and sweated Dipsaci Radix by HPLC-DAD.

CONTEXT: Dipsaci Radix is derived from the dry root of Dipsacus asper Wall.ex Henry (Dipsacaceae). It has attracted increasing attention as one of the most popular and precious herbal medicines in clinical use. OBJECTIVE: To develop a HPLC-DAD method for quantitative analysis and quality control of eight active components in crude and sweated Dipsaci Radix. MATERIALS AND METHODS: The eight components in Dipsaci Radix were analyzed by HPLC-DAD on an Agilent Eclipse XDB-C18 column within a gradient elution of acetonitrile and 0.05% formic acid aqueous solution. ESI-MS spectra were acquired on a triple quadrupole mass spectrometer. Validation was performed in order to demonstrate linearity, precision, repeatability, stability, and accuracy of the method. The results were processed with principal component analysis (PCA) and discriminant analysis (DA). RESULTS: The eight components showed good linearity (R2 > 0.9991) in the ranges of 60.40-1208.00, 151.00-3020.00, 3.06-61.20, 30.76-615.20, 5.13-102.60, 10.17-203.40, 10.20-204.00, and 151.60-3032.00 mg/mL, respectively. The overall recoveries were in the range of 99.03-102.38%, with RSDs ranging from 1.89% to 4.05%. Through PCA, the degree of importance of the eight components in sequence was CA > AVI > IA > LA > LN > IC > IB > CaA. The crude and sweated Dipsaci Radix were distinguished obviously by DA. DISCUSSION AND CONCLUSION: The method, using HPLC-DAD analysis in combination with PCA and DA, could provide a more comprehensive and quantitative chemical pattern recognition and quality evaluation to crude and sweated Dipsaci Radix.

[Application of gut microbiota in research of Chinese medicines].

This article summarizes the research progress in recent years on interactions between Chinese medicines and gut microbiota based on the physiological functions of gut microbiota, including imbalance impacts of toxic/irritating Chinese medicines on gut microbiota, prognosis effects of Chinese medicines on gut microbiota imbalance, metabolism effects of gut microbiota on Chinese medicine components, and co-metabolism effects between gut microbiota and host. We would think and prospect the specific biological effects of Chinese medicines, gut microbiota structures and the relations between endogenous metabolites from "gut microbiota and host co-metabolism". All of these aim to investigate biological mechanisms and effective components of Chinese medicines based on gut microbiota and offer a new strategy for promoting safe and effective application of Chinese medicines.

[Rapid identification of crude and sweated dipsaci radix based on near-infrared spectroscopy combined with principal component analysis-mahalanobis distance].

In order to discriminate the crude and sweated Dipsaci Radix correctly and rapidly, the crude and sweated Dipsaci Radix were scanned by the NIR spectrometer, and an identifying model was developed by near infrared spectroscopy combined with principal component-Mahalanobis distance pattern recognition method. The pretreated spectra data of 129 crude samples and 86 sweated ones were analyzed through principal component analysis (PCA). The identifying model was developed by choosing the spectrum for 9 881.46-4 119.20 cm(-1) and "SNV + spectrum + S-G" to the original spectral preprocessing with 14 principal components, and then was verified by prediction set, identifying with 100% accuracy. The rapid identification model of the crude and sweated Dipsaci Radix by NIR is feasible and efficient, and could be used as an assistant means for identifying the crude and sweated Dipsaci Radix.